Liposomal apparatus and manufacturing methods

ABSTRACT

The present invention provides apparatus and processes for producing liposomes. By providing a buffer solution in a first reservoir, and a lipid solution in a second reservoir, continuously diluting the lipid solution with the buffer solution in a mixing chamber produces a liposome. The lipid solution preferably comprises an organic solvent, such as a lower alkanol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/203,220, filed Mar. 16, 2021, which is a continuation of U.S.application Ser. No. 16/576,587, filed Sep. 19, 2019, which is acontinuation of U.S. application Ser. No. 16/035,144, filed Jul. 13,2018, which is a continuation of U.S. application Ser. No. 15/299,413,filed Oct. 20, 2016, which is a continuation of U.S. application Ser.No. 14/304,578, filed Jun. 13, 2014, and which issued on Nov. 29, 2016as U.S. Pat. No. 9,504,651, which is a continuation of U.S. applicationSer. No. 13/684,066, filed Nov. 21, 2012, and which issued on Nov. 15,2016 as U.S. Pat. No. 9,492,386, which is a continuation of U.S.application Ser. No. 12/965,555, filed Dec. 10, 2010, and which issuedon Dec. 11, 2012 as U.S. Pat. No. 8,329,070, which is a divisional ofU.S. application Ser. No. 10/611,274, filed Jun. 30, 2003, and whichissued on Mar. 8, 2011 as U.S. Pat. No. 7,901,708, which applicationclaims priority to U.S. Provisional Application No. 60/392,887, filedJun. 28, 2002, the disclosures of which are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Many systems for administering active substances into cells are alreadyknown, such as liposomes, nanoparticles, polymer particles, immuno- andligand-complexes and cyclodextrins (see, Drug Transport in antimicrobialand anticancer chemotherapy. G. Papadakou Ed., CRC Press, 1995).Liposomes are typically prepared in the laboratory by sonication,detergent dialysis, ethanol injection or dilution, French pressextrusion, ether infusion, and reverse phase evaporation. Liposomes withmultiple bilayers are known as multilamellar lipid vesicles (MLVs). MLVsare candidates for time release drugs because the fluids entrappedbetween layers are only released as each membrane degrades. Liposomeswith a single bilayer are known as unilamellar lipid vesicles (UV). UVsmay be made small (SUVs) or large (LUVs).

Some of the methods above for liposome production impose harsh orextreme conditions which can result in the denaturation of thephospholipid raw material and encapsulated drugs. In addition, thesemethods are not readily scalable for mass production of large volumes ofliposomes. Further, lipid vesicle formation by conventional ethanoldilution, involves the injection or dropwise addition of lipid in anaqueous buffer. The resulting vesicles are typically heterogenous insize and contain a mixture of unilamellar and multilamellar vesicles.

Conventional liposomes are formulated to carry therapeutic agents eithercontained within the aqueous interior space (water-soluble drugs) orpartitioned into the lipid bilayer(s) (water-insoluble drugs). Activeagents which have short half-lives in the bloodstream are particularlysuited to delivery via liposomes. Many anti-neoplastic agents, forexample, are known to have a short half-life in the bloodstream suchthat their parenteral use is not feasible. However, the use of liposomesfor site-specific delivery of active agents via the bloodstream isseverely limited by the rapid clearance of liposomes from the blood bycells of the reticuloendothelial system (RES).

U.S. Pat. No. 5,478,860, which issued to Wheeler et al., on Dec. 26,1995, and which is incorporated herein by reference, disclosesmicroemulsion compositions for the delivery of hydrophobic compounds.Such compositions have a variety of uses. In one embodiment, thehydrophobic compounds are therapeutic agents including drugs. The patentalso discloses methods for in vitro and in vivo delivery of hydrophobiccompounds to cells. PCT Publication WO01/05373 to Knopov, et al., whichis incorporated by reference herein, discloses techniques for preparinglipid vesicles using an ethanol injection-type process with a staticmixer that provides a turbulent environment (e.g., Reynoldsnumbers >2000). Therapeutic agents may then be loaded after vesicleformation

Despite the apparent advances of U.S. Pat. No. 5,478,860 and WO05373,there exists a need for processes and apparatus for formulating andproducing lipid vesicles, and in particular lipid vesicles encapsulatinga therapeutic agent such as nucleic acid. The present invention fulfillsthese and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides processes and apparatus for making lipidvesicles that optionally contain a therapeutic agent. The therapeuticagent can include, for example, a protein, a nucleic acid, an antisensenucleic acid, a drug, or the like. The present invention can be used toform lipid vesicles that contain encapsulated plasmid DNA or smallmolecule drugs. In one aspect, the lipid vesicles are prepared rapidlyat low pressure and the approach is fully scalable. In certain preferredembodiments, the process does not involve a static mixer or specializedextrusion equipment.

As such, in one embodiment, the present invention provides a process forproducing a liposome. The process typically includes providing anaqueous solution in a first reservoir, the first reservoir in fluidcommunication with an organic lipid solution in a second reservoir, andmixing the aqueous solution with the organic lipid solution, wherein theorganic lipid solution undergoes a continuous stepwise dilution toproduce a liposome.

In certain aspects, the aqueous solution such as a buffer, comprises atherapeutic product, such that the therapeutic product is encapsulatedin the liposome. Suitable therapeutic products include, but are notlimited to, a protein, a nucleic acid, an antisense nucleic acid, aribozyme, tRNA, snRNA, siRNA (small interfering RNA), pre-condensed DNA,and an antigen. In certain preferred aspects, the therapeutic product isnucleic acid.

In another embodiment, the present invention provides a process forproducing a liposome encapsulating a therapeutic product. The processtypically includes providing an aqueous solution in a first reservoir,and providing an organic lipid solution in a second reservoir, whereinone of the aqueous solution and the organic lipid solution includes atherapeutic product. The process also typically includes mixing theaqueous solution with the organic lipid solution, wherein the organiclipid solution mixes with the aqueous solution so as to substantiallyinstantaneously produce a liposome encapsulating the therapeuticproduct. In certain aspects, the therapeutic product is a nucleic acidincluded in the aqueous solution. In certain aspects, the therapeuticproduct is lipophilic and is included in the organic lipid solution. Incertain aspects, the initial therapeutic product encapsulationefficiency is as high as about 90%.

In still yet another embodiment, the present invention providesapparatus for producing a liposome encapsulating a therapeutic product.The apparatus typically includes a first reservoir for holding anaqueous solution, and a second reservoir for holding an organic lipidsolution, wherein one of the aqueous solution and the organic lipidsolution includes a therapeutic product. the apparatus also typicallyincludes a pump mechanism configured to pump the aqueous and the organiclipid solutions into a mixing region at substantially equal flow rates.In operation, the organic lipid solution mixes with the aqueous solutionin the mixing region to substantially instantaneously form a therapeuticproduct encapsulated liposome.

These and other aspects will be more apparent when read with theaccompanying drawings and detailed descriptions that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a flow diagram for a manufacturing process according toone embodiment of the present invention.

FIG. 2 provides a schematic of a process of making liposomes in oneembodiment of the present invention.

FIG. 3 provides a schematic of an apparatus according to one embodimentof the present invention.

FIG. 4 provides a schematic of an apparatus having an ultrafiltrationsystem according to one embodiment of the present invention.

FIG. 5 shows the effect of varying the ethanol concentration of theinitial lipid solution on SPLP mean diameter and DNA encapsulation. DNAencapsulation efficiency and vesicle sizes determined after the dilutionstep.

FIG. 6 shows the effect of varying pH of the initial plasmid solution onSPLP mean diameter and DNA encapsulation. DNA encapsulation efficiencyand vesicle sizes were determined after the dilution step.

FIG. 7 shows the effect of varying pH of the buffer used for thedilution step on pDNA encapsulation efficiency.

FIG. 8 shows the effect of varying the salt concentration of the bufferused for the dilution step on pDNA encapsulation efficiency.

FIGS. 9A and 9B show a schematic process of making liposomes of thepresent invention.

FIG. 10 shows encapsulation of safranine in certain liposomes of thepresent invention.

FIG. 11 shows a schematic process of making liposomes of the presentinvention.

FIG. 12 illustrates a comparison between one embodiment of the presentinvention and an ethanol drop method for encapsulating pDNA.

FIG. 13 shows a T-connector and associated flow dynamics according toone embodiment.

FIG. 14 shows various parameters associated with flow in the T-connectorof FIG. 13.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS I.Definitions

The term “nucleic acid” refers to a polymer containing at least twonucleotides. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose(RNA), a base, and a phosphate group. Nucleotides are linked togetherthrough the phosphate groups. “Bases” include purines and pyrimidines,which further include natural compounds adenine, thymine, guanine,cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

DNA may be in the form of antisense, plasmid DNA, parts of a plasmidDNA, pre-condensed DNA, product of a polymerase chain reaction (PCR),vectors (P1, PAC, BAC, YAC, artificial chromosomes), expressioncassettes, chimeric sequences, chromosomal DNA, or derivatives of thesegroups. RNA may be in the form of oligonucleotide RNA, tRNA (transferRNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messengerRNA), antisense RNA, siRNA (small interfering RNA), ribozymes, chimericsequences, or derivatives of these groups.

“Antisense” is a polynucleotide that interferes with the function of DNAand/or RNA. This may result in suppression of expression. Naturalnucleic acids have a phosphate backbone, artificial nucleic acids maycontain other types of backbones and bases. These include PNAs (peptidenucleic acids), phosphothionates, and other variants of the phosphatebackbone of native nucleic acids. In addition, DNA and RNA may besingle, double, triple, or quadruple stranded.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., herpes simplex virus). The polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence so long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, and the like)of the full-length or fragment are retained.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

The term “lipid” refers to a group of organic compounds that are estersof fatty acids and are characterized by being insoluble in water butsoluble in many organic solvents. They are usually divided in at leastthree classes: (1) “simple lipids” which include fats and oils as wellas waxes; (2) “compound lipids” which include phospholipids andglycolipids; (3) “derived lipids” such as steroids.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while a hydrophilic portion orients toward theaqueous phase. Amphipathic lipids are usually the major component of alipid vesicle. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphato, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids and sphingolipids. Representative examples of phospholipidsinclude, but are not limited to, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Othercompounds lacking in phosphorus, such as sphingolipid, glycosphingolipidfamilies, diacylglycerols and β-acyloxyacids, are also within the groupdesignated as amphipathic lipids. Additionally, the amphipathic lipiddescribed above can be mixed with other lipids including triglyceridesand sterols.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, andother anionic modifying groups joined to neutral lipids.

The term “cationic lipid” refers to any of a number of lipid specieswhich carry a net positive charge at a selective pH, such asphysiological pH. Such lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”) andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids are available which can be used in the presentinvention. These include, for example, LIPOFECTIN® (commerciallyavailable cationic liposomes comprising DOTMA and1,2-dioleoyl-sn-3-phosphoethanolamine (“DOPE”), from GIBCO/BRL, GrandIsland, N.Y., USA); LIPOFECTAMINE® (commercially available cationicliposomes comprisingN-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (“DOSPA”) and (“DOPE”), from GIBCO/BRL); andTRANSFECTAM® (commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (“DOGS”) in ethanol from PromegaCorp., Madison, Wis., USA). The following lipids are cationic and have apositive charge at below physiological pH: DODAP, DODMA, DMDMA and thelike.

“Lipid vesicle” refers to any lipid composition that can be used todeliver a compound including, but not limited to, liposomes, wherein anaqueous volume is encapsulated by an amphipathic lipid bilayer; orwherein the lipids coat an interior comprising a large molecularcomponent, such as a plasmid, with a reduced aqueous interior; or lipidaggregates or micelles, wherein the encapsulated component is containedwithin a relatively disordered lipid mixture.

As used herein, “lipid encapsulated” can refer to a lipid formulationwhich provides a compound with full encapsulation, partialencapsulation, or both.

As used herein, the term “SPLP” refers to a stable plasmid lipidparticle. A SPLP represents a vesicle of lipids coating an interiorcomprising a nucleic acid such as a plasmid with a reduced aqueousinterior.

II. General

The present invention provides processes and apparatus for making lipidvesicles. The processes can be used to make lipid vesicles possessing awide range of lipid components including, but not limited to, cationiclipids, anionic lipids, neutral lipids, polyethylene glycol (PEG)lipids, hydrophilic polymer lipids, fusogenic lipids and sterols.Hydrophobic actives can be incorporated into the organic solvent (e.g.,ethanol) with the lipid, and nucleic acid and hydrophilic actives can beadded to an aqueous component. In certain aspects, the processes of thepresent invention can be used in preparing microemulsions where a lipidmonolayer surrounds an oil-based core. In certain preferred aspects, theprocesses and apparatus are used in preparing lipid vesicles, orliposomes, wherein a therapeutic agent is encapsulated within a liposomecoincident with liposome formation.

III. Processes of Making

FIG. 1 is an example of a representative flow chart 100 of a method ofthe present invention. This flow chart is merely an illustration andshould not limit the scope of the claims herein. One of ordinary skillin the art will recognize other variations, modifications, andalternatives.

In one aspect, the present method provides a lipid solution 110 such asa clinical grade lipid synthesized under Good Manufacturing Practice(GMP), which is thereafter solubilized in an organic solution 120 (e.g.,ethanol). Similarly, a therapeutic product, e.g., a therapeutic activeagent such as nucleic acid 112 or other agent, is prepared under GMP.Thereafter, a therapeutic agent solution (e.g., plasmid DNA) 115containing a buffer (e.g., citrate) is mixed with a lipid solution 120solubilized in a lower alkanol to form a liposomal formulation 130. Inpreferred aspects of the present invention, the therapeutic agent is“passively entrapped” in the liposome substantially coincident withformation of the liposome. However, those of skill in the art willrealize that the processes and apparatus of the present invention areequally applicable to active entrapment or loading of the liposomesafter formation of the vesicle.

According to the processes and apparatus of the present invention, theaction of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a liposome substantially instantaneously upon mixing. As usedherein, the phrase “continuously diluting a lipid solution with a buffersolution” (and variations) generally means that the lipid solution isdiluted sufficiently rapidly in an hydration process with sufficientforce to effectuate vesicle generation. By mixing the aqueous solutionwith the organic lipid solution, the organic lipid solution undergoes acontinuous stepwise dilution in the presence of the buffer (aqueous)solution to produce a liposome.

In the processes of the present invention, the organic lipid solutionpreferably includes an organic solvent, such as a lower alkanol. In oneaspect, the liposomes are then diluted 140 with a buffer (e.g., citrate)to increase nucleic acid (e.g., plasmid) entrapment. Before sampleconcentration 160, free therapeutic agent (e.g., nucleic acid) isremoved by using, for example, an anion exchange cartridge 150. Further,by using an ultrafiltration step 170 to remove the alkanol, the sampleis concentrated (e.g., to about 0.9 mg/mL plasmid DNA), the alkanol isremoved, and the buffer is replaced with a substitute buffer (e.g., witha saline buffer) 180. Thereafter, the sample is filtered 190 and filledin vials 195. The process will now be discussed in more detail hereinbelow using the steps as set forth in FIG. 1.

1. Lipid Solubilization and Therapeutic Agent Dissolution

In one embodiment, the liposome vesicles of the present processes arestable plasmid lipid particle (i.e., SPLP) formulations. Those of skillin the art will appreciate that the following description is forillustration purposes only. The processes of the present invention areapplicable to a wide range of lipid vesicle types and sizes. These lipidvesicles include, but are not limited to, single bilayer lipid vesiclesknown as unilamellar lipid vesicles which can be made small (SUVs) orlarge (LUVs), as well as multilamellar lipid vesicles (MLVs). Furthervesicles include, micelles, lipid-nucleic acid particles, virosomes, andthe like. Those of skill in the art will know of other lipid vesiclesfor which the processes and apparatus of the present invention will besuitable.

The preferred size for liposomes made in accordance with the presentprocesses and apparatus are between about 50-550 nm in diameter. Incertain preferred aspects, the liposome preparation has a sizedistribution in which the mean size (e.g., diameter) is about 70 nm toabout 300 nm, and more preferably the mean size is less than about 200nm, such as about 150 nm or less (e.g., about 100 nm).

In certain aspects, the liposome formulation (e.g., SPLP formulation) ofthe present invention includes four lipid components: a phospholipid;cholesterol; a PEG-lipid; and a cationic lipid. In one preferred aspect,the phospholipid is DSPC, the PEG-lipid is PEG-DSG and the cationiclipid is DODMA. In one preferred aspect, the molar composition is about20:45:10:25 DSPC:Chol:PEG-DSG:DODMA. In certain embodiments, the organicsolvent concentration wherein the lipids are solubilized is about 45%v/v to about 90% v/v. In certain preferred aspects, the organic solventis a lower alkanol. Suitable lower alkanols include, but are not limitedto, methanol, ethanol, propanol, butanol, pentanol, their isomers andcombinations thereof. In one embodiment, the solvent is preferablyethanol with a volume of about 50-90% v/v. Preferably, the lipids occupya volume of about 1 mL/g to about 5 mL/g.

The lipids are solubilized 120 using for example, an overhead stirrer ata suitable temperature. In one aspect, the total lipid concentration ofthe solution is about 15.1 mg/mL (20 mM). In certain preferred aspects,the therapeutic agent (e.g., nucleic acid) is included in an aqueoussolution (e.g., buffer) and is diluted to a final concentration. In onepreferred aspect, for example, the final concentration is about 0.9mg/mL in citrate buffer, with a pH of about 4.0. In this instance, thevolume of the plasmid solution is the same as the alkanol-lipidsolution. In one embodiment, the preparation of the therapeutic agent(e.g., nucleic acid) solution is performed in a jacketed stainless steelvessel with an overhead mixer. The sample does not need to be heated tobe prepared, although in certain instances it is at the same temperatureas the lipid solution prior to lipid vesicle formation.

In one embodiment, the therapeutic agent is included in the lipidsolution. In certain preferred aspects, the therapeutic agent in thelipid solution is lipophilic. Suitable lipophilic agents include taxol,taxol derivatives, including, for example, protax III and paclitaxol,lipophilic benzoporphyrins, verteporfin the lipid prodrug of foscarnet,1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA),dioleoyl[3H]iododeoxyuridine ([3H]IDU-012), lipid derivatized HIVprotease inhibitory peptides such asiBOC-[L-Phe]-[D-beta-Nal]-Pip-[alpha-(OH)-Leu]-Val (7194) and otherlipid derivatized drugs or prodrugs.

2. Liposome Formation

After the solutions, e.g., lipid solution 120 and aqueous therapeuticagent (e.g., nucleic acid) solution 115, have been prepared, they aremixed together 130 using, for example, a peristaltic pump mixer. In oneaspect, the solutions are pumped at substantially equal flow rates intoa mixing environment. In certain aspects, the mixing environmentincludes a “T”-connector or mixing chamber. In this instance, it ispreferred that the fluid lines, and hence fluid flows, meet in a narrowaperture within the “T”-connector as opposing flows at approximately180° relative to each other. Other relative introduction angles may beused, such as for example between 27° and 90° and between 90° and 180°.Upon meeting and mixing of the solution flows in the mixing environment,lipid vesicles are substantially instantaneously formed. Lipid vesiclesare formed when an organic solution including dissolved lipid and anaqueous solution (e.g., buffer) are simultaneously and continuouslymixed. Advantageously, and surprisingly, by mixing the aqueous solutionwith the organic lipid solution, the organic lipid solution undergoes acontinuous stepwise dilution to substantially instantaneously produce aliposome. The pump mechanism can be configured to provide equivalent ordifferent flow rates of the lipid and aqueous solutions into the mixingenvironment which creates lipid vesicles in a high alkanol environment.

Advantageously, and surprisingly, the processes and apparatus for mixingof the lipid solution and the aqueous solution as taught herein providesfor encapsulation of therapeutic agent in the formed liposomesubstantially coincident with liposome formation with an encapsulationefficiency of up to about 90%. Further processing steps as discussedherein can be used to further refine the encapsulation efficiency andconcentration if desired.

In one preferred aspect, using the processes and apparatus of thepresent invention, it is possible to form lipid vesicles instantaneouslyin a continuous two-step process that is fully scaleable. In one aspect,lipid vesicles are formed having a mean diameter of less than about 200nm, which do not require further size reduction by high-energy processessuch as membrane extrusion, sonication or microfluidization.

In one embodiment, lipid vesicles form when lipids dissolved in anorganic solvent (e.g., ethanol) are diluted in a stepwise manner bymixing with an aqueous solution (e.g., buffer). This controlled stepwisedilution is achieved by mixing the aqueous and lipid streams together inan aperture, such as a T-connector. The resultant lipid, solvent andsolute concentrations can be kept constant throughout the vesicleformation process.

One embodiment of the inventive process is shown in FIG. 2. In oneaspect, using the processes of the present invention, a vesicle isprepared by a two-stage step-wise dilution without gradients. Forexample, in the first stepwise dilution, vesicles are formed in a highalkanol (e.g., ethanol) environment (e.g., about 30% to about 50% v/vethanol). These vesicles can then be stabilized by lowering the alkanol(e.g., ethanol) concentration to less than or equal to about 25% v/v,such as about 17% v/v to about 25% v/v, in a stepwise manner. Inpreferred aspects, with therapeutic agent present in the aqueoussolution, or in the lipid solution, the therapeutic agent isencapsulated coincident with liposome formation.

As shown in FIG. 2, in one embodiment, lipids are initially dissolved inan alkanol environment of about 40% v/v to about 90% v/v, morepreferably about 65% v/v to about 90% v/v, and most preferably about 80%v/v to about 90% v/v (A). Next, the lipid solution is diluted stepwiseby mixing with an aqueous solution resulting in the formation ofvesicles at an alkanol (e.g., ethanol) concentration of between about37.5-50% (B). By mixing the aqueous solution with the organic lipidsolution, the organic lipid solution undergoes a continuous stepwisedilution to produce a liposome. Further, lipid vesicles such as SPLPs (alipid-particle) can be further stabilized by an additional stepwisedilution of the vesicles to an alkanol concentration of less than orequal to about 25%, preferably between about 19-25% (C).

In certain aspects, for both stepwise dilutions (A and BC), theresulting ethanol, lipid and solute concentrations are kept at constantlevels in the receiving vessel. At these higher ethanol concentrationsfollowing the initial mixing step, the rearrangement of lipid monomersinto bilayers proceeds in a more orderly fashion compared to vesiclesthat are formed by dilution at lower ethanol concentrations. Withoutbeing bound by any particular theory, it is believed that these higherethanol concentrations promote the association of nucleic acid withcationic lipids in the bilayers. In one preferred aspect, nucleic acidencapsulation occurs within a range of alkanol (e.g., ethanol)concentrations above 22%.

In certain aspects, after the lipid vesicles are formed, they arecollected in another vessel, for example, a stainless steel vessel. Inone aspect, the lipid vesicles are formed at a rate of about 60 to about80 mL/min. In one aspect, after the mixing step 130, the lipidconcentration is about 1-10 mg/mL and the therapeutic agent (e.g.,plasmid DNA) concentration is about 0.1-3 mg/mL. In certain preferredaspects, the lipid concentration is about 7.0 mg/mL and the therapeuticagent (e.g., plasmid DNA) concentration is about 0.4 mg/mL to give aDNA:lipid ratio of about 0.06 mg/mg. The buffer concentration is about1-3 mM and the alkanol concentration is about 45% v/v to about 90% v/v.In preferred aspects, the buffer concentration is about 3 mM and thealkanol concentration is about 45% v/v to about 60% v/v.

3. Liposome Dilution

Turning back to FIG. 1, after the mixing step 130, the degree oftherapeutic agent (e.g., nucleic acid) encapsulation can be enhanced ifthe lipid vesicle suspension is optionally diluted 140 prior to removalof free plasmid. For example, prior to dilution step 140, if thetherapeutic agent entrapment is at about 30-40%, it can be increased toabout 70-80% following incubation after the dilution step 140. In step140, the liposome formulation is diluted to about 10% to about 40%,preferably about 20% alkanol, by mixing with an aqueous solution such asa buffer (e.g., 1:1 with citrate buffer, 100 mM NaCl, pH 4.0). Suchfurther dilution is preferably accomplished with a buffer. In certainaspects, such further diluting the liposome solution is a continuousstepwise dilution. The diluted sample is then optionally allowed toincubate at room temperature.

4. Removal of Free Therapeutic Agent

After the optional dilution step 140, about 70-80% or more of thetherapeutic agent (e.g., nucleic acid) is entrapped within the lipidvesicle (e.g., SPLP) and the free therapeutic agent can be removed fromthe formulation 150. In certain aspects, anion exchange chromatographyis used. Advantageously, the use of an anion exchange resin results in ahigh dynamic nucleic acid removal capacity, is capable of single use,may be pre-sterilized and validated, and is fully scaleable. Inaddition, the method preferably results in removal of free therapeuticagent (e.g., nucleic acid such as approximately 25% of total plasmid).The volume of sample after chromatography is unchanged, and thetherapeutic agent (e.g., nucleic acid) and lipid concentrations areabout 0.64 and 14.4 mg/mL, respectively. At this point, the sample canbe assayed for encapsulated therapeutic agent and adjusted to about 0.55mg/mL.

5. Sample Concentration

In certain instances, the liposome solution is optionally concentratedabout 2-6 fold, preferably about 4 fold, using for example,ultrafiltration 160 (e.g., tangential flow dialysis). In one embodiment,the sample is transferred to a feed reservoir of an ultrafiltrationsystem and the buffer is removed. The buffer can be removed usingvarious processes, such as by ultrafiltration. In one aspect, buffer isremoved using cartridges packed with polysulfone hollow fibers, forexample, having internal diameters of about 0.5 mm and a 30,000 nominalmolecular weight cut-off (NMWC). The liposomes are retained within thehollow fibers and recirculated while the solvent and small molecules areremoved from the formulation by passing through the pores of the hollowfibers. In this procedure, the filtrate is known as the permeatesolution. On completion of the concentration step, the therapeutic agent(e.g., nucleic acid) and lipid concentrations increase to about 0.90 and15.14 mg/mL, respectively. In one embodiment, the alkanol concentrationremains unchanged, but the alkanol:lipid ratio decreases about fourfold.

6. Alkanol Removal

In one embodiment, the concentrated formulation is then diafiltratedagainst about 5-15 volumes, preferably about 10 volumes, of aqueoussolution (e.g., buffer) (e.g., citrate buffer pH 4.0 (25 mM citrate, 100mM NaCl) to remove the alkanol 170. The alkanol concentration at thecompletion of step 170 is less than about 1%. Preferably, lipid andtherapeutic agent (e.g., nucleic acid) concentrations remain unchangedand the level of therapeutic agent entrapment also remains constant.

7. Buffer Replacement

After the alkanol has been removed, the aqueous solution (e.g., buffer)is then replaced by dialfiltration against another buffer 180 (e.g.,against 10 volumes of saline 150 mM NaCl with 10 mM Hepes pH 7.4).Preferably, the ratio of concentrations of lipid to therapeutic agent(e.g., nucleic acid) remain unchanged and the level of nucleic acidentrapment is about constant. In certain instances, sample yield can beimproved by rinsing the cartridge with buffer at about 10% volume of theconcentrated sample. In certain aspects, this rinse is then added to theconcentrated sample.

8. Sterile Filtration

In certain preferred embodiments, sterile filtration 190 of the sampleat lipid concentrations of about 12-14 mg/mL can optionally beperformed. In certain aspects, filtration is conducted at pressuresbelow about 40 psi, using a capsule filter and a pressurized dispensingvessel with a heating jacket. Heating the sample slightly can improvethe ease of filtration.

9. Sterile Fill

The sterile fill step 195 is performed using similar processes as forconventional liposomal formulations. The processes of the presentinvention result in about 50-60% of the input therapeutic agent (e.g.,nucleic acid) in the final product. In certain preferred aspects, thetherapeutic agent to lipid ratio of the final product is approximately0.04 to 0.07.

IV. Therapeutic Agents

The lipid-based drug formulations and compositions of the presentinvention are useful for the systemic or local delivery of therapeuticagents or bioactive agents and are also useful in diagnostic assays. Thefollowing discussion refers generally to liposomes; however, it will bereadily apparent to those of skill in the art that this same discussionis fully applicable to the other drug delivery systems of the presentinvention.

As described above, therapeutic agent is preferably incorporated intothe lipid vesicle during formation of the vesicle. In one embodiment,hydrophobic actives can be incorporated into the organic solvent withthe lipid, while nucleic acid and hydrophilic actives can be added tothe aqueous component. In certain instances, the therapeutic agentincludes one of a protein, a nucleic acid, an antisense nucleic acid,ribozymes, tRNA, snRNA, siRNA, pre-condensed DNA, an antigen andcombinations thereof. In preferred aspects, the therapeutic agent isnucleic acid. The nucleic acid may encode a protein such as, forexample, a herpes simplex virus, thymidine kinase (HSV-TK), a cytosinedeaminase, a xanthine-guaninephosphoribosyl transferase, a p53, a purinenucleoside phosphorylase, a carboxylesterase, a deoxycytidine kinase, anitroreductase, a thymidine phosphorylase, or cytochrome P450 2B1.

In certain aspects, therapeutic agent is incorporated into the organiclipid component. In certain instances, the therapeutic agent islipophilic. Suitable lipophilic agents include taxol, taxol derivatives,including, for example, protax III and Paclitaxol, lipophilicbenzoporphyrins, verteporfin the lipid prodrug of foscarnet,1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA),dioleoyl[3H]iododeoxyuridine ([3H]IDU-012), lipid derivatized HIVprotease inhibitory peptides such asiBOC-[L-Phe]-[D-beta-Nal]-Pip-[alpha-(OH)-Leu]-Val (7194) and otherlipid derivatized drugs or prodrugs.

In another embodiment, the lipid vesicles of the present invention canbe loaded with one or more therapeutic agents after formation of thevesicle. In certain aspects, the therapeutic agents which areadministered using the present invention can be any of a variety ofdrugs which are selected to be an appropriate treatment for the diseaseto be treated. Often the drug is an antineoplastic agent, such asvincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin,bleomycin, cyclophosphamide, methotrexate, streptozotocin, and the like.Especially preferred antitumor agents include, for example, actinomycinD, vincristine, vinblastine, cystine arabinoside, anthracyclines,alkylative agents, platinum compounds, antimetabolites, and nucleosideanalogs, such as methotrexate and purine and pyrimidine analogs. It mayalso be desirable to deliver anti-infective agents to specific tissuesby the present processes. The compositions of the present invention canalso be used for the selective delivery of other drugs including, butnot limited to, local anesthetics, e.g., dibucaine and chlorpromazine;beta-adrenergic blockers, e.g., propranolol, timolol and labetolol;antihypertensive agents, e.g., clonidine and hydralazine;anti-depressants, e.g., imipramine, amitriptyline and doxepim;anti-conversants, e.g., phenytoin; antihistamines, e.g.,diphenhydramine, chlorphenirimine and promethazine;antibiotic/antibacterial agents, e.g., gentamycin, ciprofloxacin, andcefoxitin; antifungal agents, e.g., miconazole, terconazole, econazole,isoconazole, butaconazole, clotrimazole, itraconazole, nystatin,naftifine and amphotericin B; antiparasitic agents, hormones, hormoneantagonists, immunomodulators, neurotransmitter antagonists,antiglaucoma agents, vitamins, narcotics, and imaging agents.

V. Apparatus

In another embodiment, the present invention provides apparatus forcarrying out the processes of the present invention. FIG. 3 is anexample of a representative schematic of an apparatus 300 according toone embodiment of the present invention. This schematic is merely anillustration and should not limit the scope of the claims herein. One ofordinary skill in the art will recognize other variations,modifications, and alternatives.

In one embodiment, the apparatus of the present invention includes tworeservoirs, an aqueous solution reservoir 310 and an organic solutionreservoir 320, for holding aqueous solution and organic solution,respectively. In certain aspects, the lipid vesicle formulations areprepared rapidly, at low pressure (e.g., <10 psi) and the apparatus andprocesses of the present invention are fully scaleable (e.g., 0.5mL-5000 L). At a 1-L scale, lipid vesicles are formed at about 0.4-0.8L/min. In certain preferred aspects, the apparatus do not use staticmixers nor specialized extrusion equipment.

The mixing chamber 340 is, in one embodiment, a T-connector, havingoptional hose barbs, wherein fluid lines 334 and 336 impact each otherat about 180°. The angle of mixing can also be changed, and lipidvesicles less than about 100 nm can be formed at angles of between about27° and about 90° or even between 90° and 180°. In preferred aspects,lipid vesicles of well defined and reproducible mean diameters areprepared using substantially equal flow rates of the flow lines. Inother aspects, lipid vesicles of well defined and reproducible meandiameters are prepared by changing the flow rate of the fluid lines,e.g., to ensure sufficient mixing in some cases. In preferred aspects,the variance between flow rates is less than 50%, more preferably lessthan about 25% and even more preferably less than about 5%.

FIG. 13 shows a T-connector and associated flow dynamics according toone embodiment. Examples of flow rates, and resulting shear rates andReynolds numbers (turbulence measure) are shown in FIG. 14 and discussedin more detail hereafter in Example 8. In comparison with prior systems,the present invention provides non-turbulent flow and increased shearrates at much lower (and substantially equivalent) flow rates. Forexample, the present invention advantageously provides non-turbulentflow (N_(re)<2000) in the mixing environment with a shear rate betweenabout 500/s and about 3300/s at a flow rate (both flow lines) of betweenabout 0.075 and about 0.3 L/min.

Mixing of the two fluid components can be driven using, for example, aperistaltic pump 330, a positive displacement pump, or by pressurizingboth the lipid-ethanol and buffer vessels 320, 310. In one aspect, aWatson-Marlow 505 Di/L pump fitted with a 505 L pump head is used;silicone tubing (e.g., platinum cured with 3.2 mm ID, 2.4 mm wallthickness; available from Watson Marlow as catalog no. 913A032024) canbe used for flow lines into a polypropylene or stainless steelT-connector (e.g., with a ⅛″ ID). Lipid vesicles are typically formed atroom temperature, but lipid vesicles may be formed at elevatedtemperatures according to the present invention. Unlike other existingapproaches, there are no general requirements for buffer composition. Infact, the processes and apparatus of the present invention can formulatea lipid vesicle by mixing lipid in an alkanol with water. In certainaspects, the processes and apparatus of the present invention form lipidvesicles that are less than 200 nm in diameter.

When lipid vesicles are prepared containing plasmid DNA (such as SPLPs),the ratio of plasmid to cationic lipid and counter ions can beoptimized. For refined formulations, 70-95% plasmid DNA (“pDNA”)encapsulation after mixing, and ethanol removal steps is preferred. Thelevel of pDNA encapsulation can be increased by diluting this initialSPLP formulation. Surprisingly, the processes and apparatus of thepresent invention provide an encapsulation efficiency, upon mixing thesolutions (with therapeutic agent in one of the solution components) inthe mixing environment, of up to about 90%. Further refinement, e.g.,dilution, may be performed as discussed herein.

In certain aspects, liposome producing apparatus 300 of the presentinvention further includes a temperature control mechanism (not shown)for controlling the temperature of the reservoirs 310 and 320.Preferably, fluid from the first reservoir 310 and the second reservoirs320 flows into mixing chamber 340 simultaneously at separate apertures.Apparatus 300 further includes a collection reservoir 350 downstream ofthe mixing chamber for liposome collection. Moreover, in certainaspects, apparatus 300 further includes storage vessels upstream ofeither or both of the reservoirs 310 and 320. Further, either or both ofthe reservoirs 310 and 320 are preferably jacketed stainless steelvessels equipped with an overhead mixer.

In another embodiment, the present invention provides an apparatushaving an ultrafiltration system for carrying out the processes of thepresent invention. FIG. 4 is an example of a representative schematic ofan apparatus 400 according to one embodiment of the present invention.This schematic is merely an illustration and should not limit the scopeof the claims herein. One of ordinary skill in the art will recognizeother variations, modifications, and alternatives.

In certain aspects, apparatus 400 includes a plurality of reservoirs andis equipped with an ultrafiltration system. An aqueous solutionreservoir 440 and an organic solution reservoir 430 each have upstreampreparation vesicles 433 and 425, respectively. In one aspect, lipidpreparation vessel 425 is optionally equipped with an alkanol storagevessel 421 in fluid communication therewith.

As shown in FIG. 4, the ultrafiltration system includes an incubationvessel 450 in fluid communication with a collection vessel 455, anexchange column 460 and a tangential flow ultrafiltration cartridge 465.The ultrafiltration system optionally includes a permeate vessel 470. Incertain aspects, ultrafiltration is used to concentrate SPLP samples andthen remove ethanol from the formulation by buffer replacement.

In one embodiment of operation, the diluted SPLPs are transferred to thefeed reservoir of the ultrafiltration system. Concentration is performedby removing buffer and ethanol using, for example, cross flow cartridges465 packed with polysulfone hollow fibers that possess internaldiameters of about 0.5 mm and a 100,000 molecular weight cut-off (MWCO).The SPLPs are retained within the hollow fibers and re-circulated,whereas the ethanol and buffer components are removed from theformulation by passing through the pores of these hollow fibers. Thisfiltrate is known as the permeate solution and is discarded via vessel470. After the SPLPs are concentrated to the desired plasmidconcentration, the buffer in which the SPLPs are suspended is removed byultrafiltration and replaced by an equal volume of the final buffer.Ultrafiltration can be replaced with other methods such as conventionaldialysis.

VI. Examples Example 1

This Example illustrates various physical and chemical properties ofSPLPs made in accordance with one embodiment of the present invention.

Table I the amount of ethanol, pDNA and lipid content in process stepsaccording to the present invention.

TABLE I % pDNA Lipid Initial [Ethanol] [pDNA] Recovery [Lipid] RecoverySTEP Volume (%) (mg/ml) (%) (mg/ml) (%) SPLP 100 45   0.45 95 7.6 95formation Dilution 200 22.5 0.23 90 3.8 90 Concentration  50 22.5 0.9090 15.1  90 Ethanol  50   <1% 0.90 90 15.1  90 removal Buffer  45 <0.1%0.90 81 15.1  81 replacement* Free DNA  45 <0.1% 0.64 55 14.4  76Removal** (0.55) (12.4)  Sterile  49 <0.1% 0.50 50 11.1  68 filtration &Vial fill*** *Estimate 10% total volume and SPLP loss after bufferreplacement step. **Assume that 75% of pDNA is encapsulated and all freeDNA is removed. Estimate 5% loss of SPLP on anion exchange cartridge. Atthis step the sample will be assayed for encapsulated pDNA and adjustedto 0.55 mg/ml to anticipate loss of SPLP during the filtration step(concentrations after adjustment to 0.55 mg/ml pDNA shown in brackets).***Assume a maximum 5% volume loss and up to 10% total SPLP loss.

Table II sets forth the plasmid specification made according to oneaspect of the present invention.

TABLE II Plasmid Specification Test Specification 1. Appearance Clear,Colorless solution. 2. Electrophoresis Relative migration vs standard.3. Circular plasmid >90% 4. Potentiometric pH 6.5-8.5 value 5.Electrophoresis RNA undetectable 6. BCA protein assay Undetectable 7.Spectrometric 1.7-2.0 A₂₆₀/A₂₈₀ 8. DNA hybridization <1% E. coli DNAassay 9. Sterility Testing No growth observed in bacteriologic media 10.LAL <20 EU/mg. 11. UV Absorbance 2.0-3.0 mg/mL.

Table III sets forth the SPLP specification made according to one aspectof the present invention.

TABLE III Test Specification 1. Appearance Homogenous, opaque whitesolution 2. pH 7.4 (6.0-8.5) 3. Osmolality 320 mOsm/kg (290-500 mOsm/kg)4. Plasmid Content 0.5 mg/mL (0.25- 1.0 mg/mL) 5. DSPC Content 20 +/−4.0 mol % 6. DODMA Content 25 +/− 5.0 mol % 7. PEG-DSG Content 10 +/−2.0 mol % 8. Cholesterol Content 45 +/− 5.0 mol % 9. Particle size Meandiameter 100 ± 25 nm 10. Plasmid Encapsulation >85% 11. PlasmidIntegrity Supercoiled >80% Nicked <20% Linear  <2% 12. LAL <50 EU/mg DNA13. Sterility Pass

Example 2

This Example illustrates various process parameters in one embodiment ofthe present invention.

In one SPLP embodiment, varying the initial ethanol concentration forlipid dissolution had little impact on either vesicle size or DNAencapsulation, providing that the ethanol concentration was high enoughto ensure that none of the individual lipid components precipitated(see, FIG. 5). Below 75% ethanol, lipids were not soluble even withheating to 55° C. Lipids dissolved in 75% ethanol at 55° C. formed SPLPwith larger mean diameters and lower DNA encapsulation (see, FIG. 5).

The initial DNA to lipid ratio has been varied from 0.048-0.081 mg DNA:mg lipid formulation and vesicles of similar size with 77-90% DNAencapsulation were formed.

SPLPs have been prepared at a pH range of about 3.5-6 for the initialmixing step and all formulations possessed mean particle diameters ofless than 150 nm and DNA encapsulation efficiencies of greater than 50%(see, FIG. 6). At higher pH, vesicles can also be prepared with similarvesicle sizes, but with lower DNA encapsulation efficiencies.

In certain aspects, mean vesicle diameters of empty vesicles preparedusing one process of the present invention depend upon the saltconcentration of the diluting buffer, (e.g.,Sphingomyelin:cholestesterol vesicles, EPC:EPG vesicles). Varying theionic conditions in the buffer, influences the tendency for a givenlipid to arrange itself into bilayers and vesicles.

During the development of one SPLP formulation, it was found that boththe pH and salt concentration of the diluting buffer had a significanteffect on the DNA encapsulation efficiency. Naturally, diluting bufferswith pH values lower than the pKa for the cationic lipid component(DODMA) gave higher encapsulation values (FIG. 7). Interestingly, afinal salt concentration of 150 mM was also optimal for DNAencapsulation (FIG. 8).

Example 3

This Example illustrates the use of one process of the present inventionto make EPC and POPC vesicles.

POPC vesicles are useful as “sink” vesicles for membrane fusion assays.In particular, they can be used in excess to remove PEG lipids fromother liposomes, thus destabilizing the other liposomes and allowingthem to fuse with the desired membrane. EPC vesicles are useful forremoving cholesterol from arterial plaques.

The vesicles were prepared at an initial ethanol concentration of 80%,and lipid concentration of 10 mM. After mixing and dilution, the ethanolconcentration was 20%, and lipid concentration was 5 mM. The EPCformulation was mixed and diluted with PBS, and the POPC was mixed anddiluted with HBS. Both preparations were concentrated and ethanolremoved using an ultrafiltration cartridge, i.e., the EPC against PBS,and the POPC against HBS. Both preparations were then sterile filteredusing 0.22 um syringe filters.

TABLE IV EPC and POPC Vesicle Data Vesicle Size (nm) Lipid ConcentrationSample Lot Number Diam SD Chi² mg/mL POPC 25031302-02 125 62 7 22.0 EPC25031302-01 89 39 9 18.2

Example 4

This Example illustrates the use of one process of the present inventionto make EPC/Cholesterol vesicles with a pH gradient.

Unilamellar lipid vesicles (LUV) comprising EPC and Cholesterol havetraditionally been prepared by hydrating lipid films to formmultilamellar lipid vesicles (MLV) that have been subjected to vesiclesize reduction using high-pressure extrusion. It is well known thatthese vesicles can be prepared with acidic aqueous interiors and a pHgradient across the lipid bilayer. Weakly basic lipophilic moleculeshave been shown to accumulate in these vesicles at high internalconcentrations. Various drug-loaded liposomes that are currently in latestage clinical trials utilize this approach (e.g., Myocet: doxorubicinloaded vesicles).

In one aspect, safranine was used to determine whether such a pHgradient was present. Safranine is a lipophilic basic dye that has beenused to study membrane pH gradients

EPC/Chol vesicles were prepared using the present processes andapparatus at an initial ethanol concentration of 80%, and lipidconcentration of 10 mM (See FIG. 9A-B). After mixing and dilution, theethanol concentration was 20%, and lipid concentration was 5 mM. Threedifferent formulations were prepared:

1. Mixed and diluted with PBS (control).

2. Mixed and diluted with 150 mM citrate (final citrate concentration is94 mM).

3. Mixed and diluted with 300 mM citrate (final citrate concentration is188 mM).

After mixing and dilution, each sample was concentrated and ethanol wasremoved using ultrafiltration. After the concentration step, each samplewas diafiltrated against its diluting buffer to ensure that the acidiccitrate buffer present within vesicles would not leak out during ethanolremoval. All samples were finally formulated with an external buffer ofphosphate-buffered saline at pH 7.4. After sterile filtration, the meanvesicle diameters of these formulations were very similar (90-92 nm) andpossessed acceptable standard deviation and Chi squared values (TableV).

Following dialysis, the vesicles were assayed for lipid concentrationusing the Infinity cholesterol assay. Solutions were then preparedcontaining 5 mM lipid and 0.2 mM safranine obtained from a filtered 10mM stock solution. The solutions were incubated at 37° C. for 30minutes. A 500 ul aliquot of each incubated solution was then passeddown a 2-mL Sepharose CL4B gel filtration column. The free dye wasseparated from the vesicles, and the lipid-containing fractions werecollected and analyzed. The safranine concentration was determined bymeasuring the fluorescence of the samples at 516 nm excitation and 585nm emission.

The vesicles with acidic interiors accumulated safranine, with the 94 mMcitrate-containing vesicles showing the highest encapsulation. Incontrast, the PBS control vesicles encapsulated very little safranine.The 188 mM citrate vesicles also encapsulated some safranine, but not asmuch as the 94 mM citrate-containing vesicles (See FIG. 10).

TABLE V Safranine-Loaded EPC/Chol Vesicles Safranine Vesicle Size (nm)Dye: Lipid Ratio Sample Encapsulation Diam SD Chi² Mg:mg mol:mol PBSControl  9% 90 33 2.7 0.002 0.003  94 mM Citrate 54% 92 41 1.7 0.0110.019 188 mM Citrate 31% 91 35 4.8 0.007 0.011

Example 5

This Example illustrates the use of one process of the present inventionto make sphingomyelin/cholesterol vesicles.

Sphingomyelin/cholesterol vesicles are desirable due to their durabilityand strength. These vesicles can also be used to encapsulate drugs usinga pH gradient. However, these LUV have traditionally needed to be formedat temperatures greater than 65° C. and using high pressure extrusion.In order to form these vesicles with the lipomixer, a number ofvariables needed to be taken into consideration, such as ethanolconcentration, lipid concentration, and the salt concentration of themixing and dilution buffer.

The vesicles were formulated at a ratio of 55/45 SM/Chol (mol:mol),while the initial ethanol concentration after mixing varied from 50 to25%. Dilution buffers tested included PBS, water, 10 mM citrate, 150 mMcitrate, and 300 mM citrate. Final lipid concentrations ranged from 0.5to 2.5 mM. The vesicles formulated in the presence of salt (i.e., usingbuffers) were 200-500 nm, indicating an MLV. Aliquots of these sampleswere dialyzed against both 150 mM citrate and water in an attempt toremove ethanol and stabilize the vesicles.

Example 6

This Example illustrates the use of one process of the present inventionto prepare vesicles that passively encapsulation small molecules such ascalcein.

Calcein is a fluorescent dye that is self-quenching at concentrationsgreater than 10 mM. Vesicles encapsulating calcein can be used in fusionassays to determine whether vesicles have fused together. Fusiondecreases the internal calcein concentration, causing it to fluoresce.Vesicles were prepared with DSPC:CHOL:PEG-DLG:DODMA (20:55:10:15) at anethanol concentration of 19% and 2 mM lipid after mixing and dilution(See FIG. 11). Lipids dissolved in ethanol were mixed with a solutioncontaining 20 mM citrate and 75 mM calcein, and then the resultingvesicles were diluted with 300 mM NaCl and 37.5 mM Calcein. The calceinwas obtained from a 100 mM stock solution. The final calceinconcentration in the vesicles was 37.5 mM.

After mixing and dilution the vesicles were dialyzed overnight againstFIBS to remove unencapsulated dye. This was unsuccessful at removing allof the free dye, so the vesicles were passed down a gel filtrationcolumn. The lipid fraction was collected and analyzed. It was found thatthe calcein was indeed self quenching at the concentration inside thevesicles. This is a clear demonstration that the processes and apparatusof the present invention can be used to prepare vesicles that passivelyencapsulate small molecules.

TABLE VI Calcein-encapsulated vesicles Vesicle Size (nm) FluorescenceStep Diam SD Chi² rF_(without triton) rF_(with triton) Post Dilution 205109 0.4 N/d N/d Post Dialysis 173 74 0.5 N/d N/d Post Gel Filtration 17877 5.4 0.4 4.1

Example 7

This Example illustrates the use of one process of the present inventionversus prior art methods.

With reference to FIG. 12, lipids were dissolved in 90% ethanol (A) anddiluted either: step-wise using an apparatus of the present invention to45% (B) and 22.5% ethanol (C), represented by the solid line(“LipoMixer”); or added drop-wise with into stirred buffer to a finalethanol concentration of 22.5% (C), represented by the dotted line. Eventhough the final ethanol concentrations for both preparations were thesame, the SPLP formed according to the processes of the presentinvention had 85% DNA encapsulation whereas vesicles prepared by ethanoldrop had only 5% DNA encapsulation.

Example 8

This example illustrates various conditions and properties for formingliposomes according to the present invention. It should be appreciatedthat other conditions and parameters may be used and that those usedherein are merely exemplary.

With reference to FIGS. 13 and 14, various flow rates (substantiallyequivalent for both lipid and aqueous solution flows) are modeled andanalyzed to show various parameters such as shear rate and Reynoldsnumber (N_(re)) and vesicle size. Parameters and conditions weredetermined at the outlet of the T-connector correcting for the densityand viscosity of the resulting ethanol solution. Additional turbulenceas a result of the two streams meeting one another in opposition has notbeen accounted for, nor has additional turbulence as a result of thestreams having to turn a 90 degree corner.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes

What is claimed is:
 1. A process for producing a lipid vesicleencapsulating a nucleic acid within the lipid vesicle, the processcomprising: providing an aqueous solution including a nucleic acid in afirst reservoir; providing an organic lipid solution in a secondreservoir, wherein the lipids present in the organic lipid solution aresolubilized in a lower alkanol at a concentration of about 75% v/v to100% v/v; introducing the aqueous solution and the organic lipidsolution into a mixing chamber as opposing flows at about 180° relativeto each other and at different flow rates relative to each other; andmixing the organic lipid solution with the aqueous solution, wherein themixing produces a lipid vesicle encapsulating the nucleic acid withinthe lipid vesicle by diluting the concentration of the lower alkanol inthe organic lipid solution.
 2. The process of claim 1, wherein thelipids present in the organic lipid solution comprise a phospholipid,cholesterol, a PEG-lipid, and a cationic lipid.
 3. The process of claim2, wherein a plurality of lipid vesicles are produced and said pluralityof lipid vesicles have a mean particle diameter of less than 150 nm. 4.The process of claim 2, wherein the aqueous solution includes an acidicbuffer.
 5. The process of claim 2, wherein the aqueous solution includesa buffer having a pH lower than the pKa for the cationic lipidcomponent.
 6. The process of claim 2, wherein mixing the organic lipidsolution with the aqueous solution produces a plurality of lipidvesicles at a pH range of about 3.5 to about
 6. 7. The process of claim2, wherein mixing the organic lipid solution with the aqueous solutionproduces a plurality of lipid vesicles at a pH range of about 3.5 toabout
 8. 8. The process of claim 1, wherein the nucleic acid comprisesan RNA.
 9. The process of claim 8, wherein the lower alkanol comprisesethanol and wherein the lipids present in the organic lipid solutioncomprise a phospholipid, cholesterol, a PEG-lipid, and a cationic lipid.10. The process of claim 9, wherein the aqueous solution includes anacidic buffer.
 11. The process of claim 9, wherein the aqueous solutionincludes a buffer having a pH lower than the pKa for the cationic lipidcomponent.
 12. The process of claim 9, wherein mixing the organic lipidsolution with the aqueous solution produces a plurality of lipidvesicles at a pH range of about 3.5 to about 6 and said plurality oflipid vesicles have a mean particle diameter of less than 150 nm. 13.The process of claim 8, wherein the RNA comprises an mRNA.
 14. Theprocess of claim 13, wherein the lower alkanol comprises ethanol andwherein the lipids present in the organic lipid solution comprise aphospholipid, cholesterol, a PEG-lipid, and a cationic lipid.
 15. Theprocess of claim 14, wherein the aqueous solution includes an acidicbuffer.
 16. The process of claim 14, wherein mixing the organic lipidsolution with the aqueous solution produces a plurality of lipidvesicles at a pH range of about 3.5 to about 6 and said plurality oflipid vesicles have a mean particle diameter of less than 150 nm. 17.The process of claim 14, wherein mixing the organic lipid solution withthe aqueous solution produces a plurality of lipid vesicles at a pHrange of about 3.5 to about
 8. 18. A process for producing a lipidvesicle encapsulating a nucleic acid within the lipid vesicle, theprocess comprising: providing an aqueous solution including, a nucleicacid in a first reservoir; providing an organic lipid solution in asecond reservoir, wherein the lipids present in the organic lipidsolution are solubilized in a lower alkanol at a concentration of about75% v/v to 100% v/v; introducing the organic lipid solution and theaqueous solution into a mixing chamber at different flow rates relativeto each other and at an angle of between 90° and 180° relative to eachother; mixing the organic lipid solution with the aqueous solution,wherein the mixing produces a lipid vesicle encapsulating the nucleicacid within the lipid vesicle by diluting the concentration of the loweralkanol in the organic lipid solution.
 19. The process of claim 18,wherein the lower alkanol comprises ethanol and wherein the lipidspresent in the organic lipid solution comprise a phospholipid,cholesterol, a PEG-lipid, and a cationic lipid.
 20. The process of claim19, wherein the aqueous solution includes an acidic buffer having a pHlower than the pKa for the cationic lipid component.
 21. The process ofclaim 20, wherein mixing the organic lipid solution with the aqueoussolution produces a plurality of lipid vesicles at a pH range of about3.5 to about 6 and said plurality of lipid vesicles have a mean particlediameter of less than 150 nm.
 22. The process of claim 20, whereinmixing the organic lipid solution with the aqueous solution produces aplurality of lipid vesicles at a pH range of about 3.5 to about
 8. 23.The process of claim 18, wherein the nucleic acid comprises an RNA. 24.The process of claim 23, wherein the lipids present in the organic lipidsolution comprise a phospholipid, cholesterol, a PEG-lipid, and acationic lipid.
 25. The process of claim 24, wherein the aqueoussolution includes an acidic buffer.
 26. The process of claim 24, whereinmixing the organic lipid solution with the aqueous solution produces aplurality of lipid vesicles at a pH range of about 3.5 to about
 8. 27.The process of claim 23, wherein the RNA comprises an mRNA.
 28. Theprocess of claim 27, wherein the lipids present in the organic lipidsolution comprise a phospholipid, cholesterol, a PEG-lipid, and acationic lipid.
 29. The process of claim 28, wherein the aqueoussolution includes a buffer having a pH lower than the pKa for thecationic lipid component.
 30. The process of claim 28, wherein mixingthe organic lipid solution with the aqueous solution produces aplurality of lipid vesicles at a pH range of about 3.5 to about 6 andsaid plurality of lipid vesicles have a mean particle diameter of lessthan 150 nm.